MicroRNA-29c-3p in Dual-Labeled Exosome is Potential Marker of Alzheimer's Disease

ObjectiveWe aimed to establish a method to determine whether peripheral blood NCAM/amphiphysin 1 dual-labeled exosomal proteins and microRNA might be serve as a marker for the diagnosis of Alzheimer's disease.MethodsIt was a multicenter study using a two-stage design. The subjects included 45 SCD, 50 aMCI, 40 AD patients, and 30 controls in the discovery stage; the results were confirmed in the verification stage (47 SCD, 45 aMCI, 45 AD, and 30 controls). Peripheral blood NCAM single-labeled and NCAM/amphiphysin 1 double-labeled exosomes were captured and detected by immunoassay, respectively.ResultsLevels of Aβ42, Aβ42/40, Tau, P-T181-tau, and miR-29c-3p in plasma NCAM single-labeled and NCAM/amphiphysin 1 double-labeled exosomes of the aMCI and AD groups were significantly higher than those of the SCD group, control group and VaD group (all P<0.05). The levels of Aβ42 and miR-29c-3p in peripheral blood NCAM/amphiphysin 1 dual-labeled exosome was higher than that in the control and VaD groups (all P<0.05). The levels of exosomal Aβ42, Aβ42/40, Tau, P-T181-tau, and miR-29c-3p in peripheral blood were correlated with that in CSF (all P<0.05). ConclusionThis study first provided a method for sorting specific surface marker exosomes using a two-step immune capture technology. The plasma NCAM/amphiphysin 1 dual-labeled exosomal Aβ42/40 and miR-29c-3p had potential advantages in the diagnosis of SCD.

Integrin-mediated adhesions in regulation of cellular senescence

Bioinformatic and functional data link integrin-mediated cell adhesion to cellular senescence; however, the significance of and molecular mechanisms behind these connections are unknown. We now report that the focal adhesion–localized βPAK-interacting exchange factor (βPIX)–G protein–coupled receptor kinase interacting protein (GIT) complex controls cellular senescence in vitro and in vivo. βPIX and GIT levels decline with age. βPIX knockdown induces cellular senescence, which was prevented by reexpression. Loss of βPIX induced calpain cleavage of the endocytic adapter amphiphysin 1 to suppress clathrin-mediated endocytosis (CME); direct competition of GIT1/2 for the calpain-binding site on paxillin mediates this effect. Decreased CME and thus integrin endocytosis induced abnormal integrin signaling, with elevated reactive oxygen species production. Blocking integrin signaling inhibited senescence in human fibroblasts and mouse lungs in vivo. These results reveal a central role for integrin signaling in cellular senescence, potentially identifying a new therapeutic direction.

Interaction of amphiphysins with AP-1 clathrin adaptors at the membrane

The assembly of clathrin/AP (adaptor protein)-1-coated vesicles on the trans-Golgi network and endosomes is much less studied than that of clathrin/AP-2 vesicles at the plasma membrane for endocytosis. In vitro, the association of AP-1 with protein-free liposomes had been shown to require phosphoinositides, Arf1 (ADP-ribosylation factor 1)–GTP and additional cytosolic factor(s). We have purified an active fraction from brain cytosol and found it to contain amphiphysin 1 and 2 and endophilin A1, three proteins known to be involved in the formation of AP-2/clathrin coats at the plasma membrane. Assays with bacterially expressed and purified proteins showed that AP-1 stabilization on liposomes depends on amphiphysin 2 or the amphiphysin 1/2 heterodimer. Activity is independent of the SH3 (Src homology 3) domain, but requires interaction of the WDLW motif with γ-adaptin. Endogenous amphiphysin in neurons and transfected protein in cell lines co-localize perinuclearly with AP-1 at the trans-Golgi network. This localization depends on interaction of clathrin and the adaptor sequence in the amphiphysins and is sensitive to brefeldin A, which inhibits Arf1-dependent AP-1 recruitment. Interaction between AP-1 and amphiphysin 1/2 in vivo was demonstrated by co-immunoprecipitation after cross-linking. These results suggest an involvement of amphiphysins not only with AP-2 at the plasma membrane, but also in AP-1/clathrin coat formation at the trans-Golgi network.